The objectives of this project are to better define growth control mechanisms in nontransformed cells, the alterations which occur in cells neoplastically transformed by chemicals and the molecular basis for these control mechanisms. The following hypotheses have been developed based on previous studies. Growth factors stimulate resting nontransformed cells to proliferate by modulating (primarily stimulating) expression of specific genes. The chemically transformed cells differ from the nontransformed cells in that they do not modulate the expression of these genes and maintain a high level of expression even at the G1 arrest state. Nontransformed cells maintained in the presence of excess growth factors behave similarly. One possible mechanism of growth factor independence of the chemically transformed cells is continual production of and response to endogenous polypeptide growth factors (transforming growth factors). A number of specific genes have been cloned, or are in the process of being cloned, whose expression is greatly induced by epidermal growth factor and/or insulin. One of these genes is related to the VL30 family of genes and another is one of the actin genes. Evidence has been derived indicating that the VL30 expression is modulated by growth factors in the nontransformed cells while the expression remains elevated in chemically transformed cells at all growth states. To study gene expression in the nontransformed and transformed cells under more carefully defined circumstances, conditions for serum-free culture of the cells are being developed. We will continue studies on the development of conditions for serum-free culture and investigation of the role of different growth factors, including transforming growth factors, on efficiently stimulating cells at different G1 arrest states to reenter the cell cycle. We will identify additional recombinant DNA clones containing sequences expressed only in growth factor-stimulated cells. Clones will be used to quantitate the expression of homologous sequences in mRNA populations under different experimental circumstances. 2-D gel electrophoresis will be used to determine the pattern of protein expression under various experimental circumstances. Selected cellular proteins will be partially purified. An understanding of the basic mechanisms involved in the control of cell proliferation and alterations which occur in neoplastic transformation may provide the key for the development of innovative means for therapy and prevention.